JPS62502662A - High level amplification and expression of foreign DNA - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] High-level amplification and expression background of foreign DNA The present invention uses endogenous adenosinase as a selectable marker for transformation and/or Encodes a protein that contains the enzyme protein in the host cell, which contains the aminodeaminase (ADA). Heterologous adenosine as a DNA coamplifier This invention relates to methods using deaminase DNA, as well as specific expression vectors.

Transformation is a process in which new genetic material does not encode the intended protein or polypeptide. Incorporation of American DNA sequences is said to be acquired by eukaryotic cells or Haratawara cells. This is a commonly used genetic manipulation method. Cell populations that typically undergo transformation The number of cells that actually take in foreign DNA is extremely small.

These problems can be solved by using selection markers in addition to exogenous DNA sequences to transform cells. You can avoid it by doing this. D where the selection marker encodes an exogenous protein The choice depends on how and how closely it is bound to the NA. Cells that carry the marker will also contain foreign DNA. use appropriate conditions By doing so, the transformed cells carrying the selection marker are prevented from taking up foreign DNA. It can be identified from the cells that are collected.

Selection is based on DNA encoding an easily identifiable marker (e.g. antibiotic resistance). involves use. Upon transformation, the cell population is tested for the presence of the marker.

Cells that have successfully taken up marker DNA are Cells that show survival (survival in medium) and fail to take up the marker are For example, they will die on contact with antibiotics.

The amount of foreign protein expressed by transformed cells is a selectable marker Similarly, when inserting DNA encoding an amplifiable gene during the transformation process, substantially increased.

Gene amplification causes the production of multiple copies of an amplifiable gene for survival. involves exposing the transformed cells to environmental pressure sufficient to Therefore, the foreign gene The use of gene amplification to achieve high levels of expression is an important technique.

The most commonly used marker/amplification system is dihydrofolate, which is present in many cell lines. The gene for reductase (DHFR) is used.

Cells transformed with DNA encoding DHPR were treated with a high concentration of methotrexe. When exposed to light (MTX), cells will amplify DHFR in order to survive. Prompted. Cells that survive this MTX selection method have a large amount of DNA encoding DHPR. have several copies. A plasmid in which the DHFR gene contains the DNA sequence of another gene When present on a host, that gene also generally becomes amplified. Therefore, When transforming cells with a vector containing the DHFR gene and a foreign gene, DHFR F'R transfers cells that have taken up the vector from cells that have not taken up the lid. DHFR acts as a selectable marker for identification, and DHFR itself is amplified, resulting in Amplify foreign DNA. DHF as a selectable and amplifiable marker The R gene has become widely used to derive transformed cell lines.

However, in reality, the DHFR system is the only cell line lacking DHFR. Its general utility is limited to the Chinese hamster ovary method (CHODHFR). Indicated. [See Urlaub (1982).゛Internal causes A cell line containing the genetic DHFR gene is one in which the endogenous DHFR is It cannot be used because it interferes with the selection of cells containing vector-containing vectors.

It was reported that when inserted into a cell line containing endogenous DHFR, A mutant DHFR gene has been reported that can be expressed. [Simo Please refer to Nson, C, C, Me. ] However, these cell lines High levels of the desired exogenous polypeptide can be obtained from transformed cells without being amplified at will. It's pretty useless when you're trying to sleep. DH in the cell body carrying the DHPR gene Exogenous proteins in this type of cell line are selectable markers that allow the use of F'H. It turned out to be difficult to obtain the optimal conditions necessary to develop goliath. .

Thus, expression and amplification of foreign proteins by the DHF'R system occurs in one cell. Limited to 5 cell lines, each cell line is always of the highest quality for the production of the intended protein. It is not necessarily a cell strain. Other cell lines also showed high CHODHFR levels under certain moxibustion conditions. CHO'DHFR- produces all of the specific protein in the cell, and also produces it better than CHO'DHFR-. will grow. Amplification and expression of heterologous DNA in a variety of different cell lines Other systems still leave unresolved problems in the field. be.

Summary of the invention One aspect of the present invention particularly provides that exogenous adenosine deaminase (ADA) ) The gene is a selectable and amplifiable matrix of cell lines containing the endogenous ADA:lt gene. It was discovered that it could be used as a marker. AI) The gene encoding Ai is Found in almost all mammalian tissues, it is essential for cell growth. It's not an enzyme. (Shipman, Cur, et al., 5cienc e200:1163-1165 (1978); Version (Hlrschor n.

R,) et al., Proc, Natl, Acad, Sci, U, S, A,, 7 3: 213-217 (1976)'. ] Therefore, the present invention A number of ADA encodes the target protein in eukaryotic cells (especially mammalian cells) allows the amplification of foreign DNA. This method uses cells containing the endogenous ADA gene. Introducing a foreign ADA gene and a heterologous gene encoding a target protein into a strain. and includes.

Cells containing the exogenous ADA gene and the heterologous protein gene are selected in their rows. genes are amplified. Finally, the heterologous protein gene is expressed and the eye The target protein is harvested.

Another aspect of the invention provides cell lines for use in ADA amplification methods. Ru. This cell line contains endogenous ADA cells with 6 ADA-encoding exogenous genes and the purpose Transformation with protein-encoding exogenous genes and co-transformation of these exogenous genes It is created by amplifying it. Then, the amplified ADA/protein gene A cell line comprising: is cultured according to the present invention. The target protein of high quality is Therefore, it is expressed. The ADA gene used in this way is human ADA or murine ADA. It can be of any known sequence of myADA. However, the tongue ADA genes in other species may be used in similar ways, depending on what the protein is used for. can do.

Yet another aspect of the invention is to coat the exogenous ADA gene and the target ring ξ protein. A novel vector containing a foreign gene for 9 is provided. These vectors are Contains polyomavirus or retrovirus sequences and may be used in the method of the present invention In order to fully transform the ADA cells or cell lines to produce the target protein, used.

Unlike the DHFR amplification system, which requires the use of DHER-cell lines, the ADA amplification method ADA-cells and ADA-cell lines only grow best under certain conditions. and/or many ADA cells and ADA cells that preferentially express the desired product. Allows for the use of stocks. It also makes the protein more effective or appropriate (e.g. (by post-translational modifications such as gamma carboxylation) It is also possible to use

Brief description of the drawing Figure 1 shows the structure of plasmid p9ADA5-29.

Figure 2 shows the structure of plasmid;'pF'VXM.

Detailed description of the invention According to the method of the present invention, a cell line containing an endogenous ADA gene is transfected with a foreign ADA cDNA. Transformed. The production of ADA cDNA is similar to the method of cloning other genes. It would be done in a similar way. [Generally known as Maniatis s, 'r,) et al., Ring Harbor Laboratory (1982); l-Wu/l/( Tool+J, J-) et al., Nature 312: 342-347 (1 984)'kgμ6. ] Human-derived ADA, cDNA and mouse-derived The base sequence of the current ADA cDNA has already been determined. [Ligington (W) igington.

D, A, et al., Nucl, Ac15 Res, 12: 1015-1024 (1984); Valerio (/Valerio, D.) et al., Gene 31 : 147-153 (1984); Yeung+C, et al., J. Bio '1. See Chem, 258:15179-15185 (1983)k. I want to be. ) ADA cDNA II'i is well known to those of ordinary skill in the art. can be incorporated into mammalian expression vectors using the developed technique f.

Cells to be transformed include yeast protoplasts and various bacterial cells. It can be any eukaryotic cell, but is preferably a cell other than a fungal cell. , most preferably a stable mammalian cell line. HeLa cells, Bowes cells Melanoma cell lines such as mouse L cells, mouse fibroblasts, mouse NEC H3T 3 and the like are useful in the method of the present invention. ADA and other genes on the chromosome Cell lines known to stably integrate into their DNA, such as Chinese ham Star ovary (CHO) line, human liver cancer Hep G2 cell line and mouse myellow cell lines may also be desirable depending on other requirements required for that cell line. .

Exogenous genes are usually not expressed in the same way as endogenous chromosomal genes. Therefore, A.D.A. Cells were transformed with exogenous ADA and the ADA gene was detected as a result of the same selection method. By undergoing amplification of The fact that transformed cells characterized by DA expression can be selected This is an interesting aspect of the present invention. Most cells produce ADA at a very low rail. ADA is unique because it is expressed in The effective introduction of expressed ADA genes is Allow for selection of transformed cells. However, a small number of ADA cell bodies, e.g. those derived from gastrointestinal and thymic tissues are produced at higher levels than those produced by most cell lines. Its use should be avoided because it produces high ADA levels. [Lee, P , A., Dev. Biol., 31: 227-233 (1973); Bar):y (Barton, R,) et al., Ce1x engineering mmuno1. .

49: 208-214 (1982); Shiji (Stat, Y.) et al., Th See ymus4: 147-154 (1982). ] Transformability The cell population exposed to are processed to identify small subpopulations that exhibit the phenotype. The cells in culture cells are screened for phenotype by applying selective pressure.

The particular selection method to be used will be determined by one of ordinary skill in the art. . Known selection methods for increased ADA expression are summarized below. skilled Researchers have adapted these and other known methods to select cells containing ectopic ADA. You will be able to do it.

One such ADA selection method involves the use of adenosine analogs. The cells are The cytotoxic adenosine analog 9-β-D-arabinofuranosyl adenine (Ara -A) i or 9-β-D-xylofuranosyl adenine (X71-A) Selected for resistance. Multi-step selection of Ara-A or X71-A inhibits ADA activity results in a strengthened cell population. [Yeung + Lila, J, Biol.

See Chem, 258 Co. 8330-8337 (1983)'. ]A DA is a combination of these adenine analogues with their respective inosine derivatives (ultimately purine). Ribose is removed by cleosibiphosphorylase to produce hypoxanthine. It has the ability to fully catalyze irreversible conversion to (detoxified by detoxification). cell become resistant to these analogs due to a deficiency in adenosine kinase activity. However, not all viable cells have increased ADA levels. right. [Chan (V, L, Chan) et al., Somatic Ce11Gen et., 7: 147-160 (1981); see the above document 1 by Yun et al. I want to be ] However, the lack of MH level of adenosine kinase is due to this adenosine It will generally be lower in cells containing diploids of kinase genes.

Method for selecting the presence of adenosine kinase [Somatic Ce1 by Chan et al. x Genetic B, 4: 1-12 (1978)] is an increase in ADA. The system has been modified to allow selection of large expression levels.

[See Yun et al., cited above, 1.5'179-15185 (1983)] . ] In contrast to the first method, all viable cells show increased ADA levels.

Adenosine kinase is activated in the presence of AAU (adenosine, aranosine/uridine) Selected. Under these growth conditions, cells are able to produce de novo AMP by alanosine. (adenosine-phosphate) biosynthesis is prevented and converts adenosine to AMP A patented adenosine kinase featuring an adenosine kinase for phosphoric acid (PRPP)'i, leading to inhibition of endogenous pyrimidine synthesis. The medium is supplemented with Uriji yi. [Green, H, et al., 5ci ence+ 182:836-837 (1973); Ishii, K.) et al., Ce'll Sci. +13:429-439 (1973). sea bream. ] However, when the concentration of adenosine increases 11 times (hereafter 1l- (abbreviated as AAU selection), high concentrations of adenosyl/ are toxic to cells, and this toxicity ADA is needed to alleviate this. [Hox (F’OX, knee H9) et al. , Ann, Rev. Biochem, 47: 655-686 (197 Please refer to 8). ] ``Once mimetic ADA is required for cell necking, the tight binding of ADA Antibiotics proven to be sexual transition state analogue inhibitors (Ka-2,5xlO) For amplification of ADA gene using high quality roteoxycoformycin (aCF)6 Please refer to ``How to select and select. ] In order to survive in these systems, most cells must requires a higher level of ADA than that produced by

When whole cells were grown in 1l-AAU conditions in the presence of increasing concentrations of aCF, , cells exhibiting high ADA expression as a result of ADA gene amplification are selected.

[Yeung+ C-+ The above documents 8333-8345 (1983)] Please refer. ] Another selection method uses deoxyadenosine as the carbon source.

By supplying cells with 2-deoxyadenosine, the cells can increase ADA activity. You can grow dependent on it. [Fernadenosine is deoxygenated by ADA It can be used as a general purine source only when converted to inosine. As a result, cells by growing in azaserine with gradually increasing concentrations of aCF. Selected for increased ADA activity. The medium should be exposed to sunlight. ] A similar method utilizing adenosine as the sole carbon source was developed by Hunt, S. W.) et al., J. Biol., Chew, 258: 13185-1319. 2.

(1983). Under these conditions, functional ADA' The required Novikoffrate hepatoma cells (Novikoffrate hepato The aCF-resistant mutant of the adeno'/7 kinase-deficient cell 2aC in medium containing adenosine as the sole carbon source with stepwise increasing F concentrations. isolated by growing. This method amplifies the ADA gene 320 times. results in thin cells. [Furthermore, Hoffθe, P, A, et al., S omatic Ce1IGenet, 8: 13185-13192 (1 983) 't-See n. ] In any cell population, a certain number of cells containing the endogenous ADA gene have higher levels of υ than other cells. ADA'i will be expressed in the cells. Therefore, the degree of selective pressure on exogenous ADA Relatively high ADA expression levels from transformed cells and the endogenous ADA gene This will affect the sensitivity of identifying the cells shown. Therefore, the endogenous ADA gene preferably at a level of five times that typically expressed by the containing cells. It is desirable to select cells that express ADAz-at a 10-fold higher level.

+ Endogenous ADA - Transformed cells exhibiting high levels of ADAI are also due to heterologous genetics. can be obtained by using vectors that provide efficient expression of the offspring. . In addition to the ADA gene and product genes, cells also contain enhancers and promoters. , intron, auxiliary DNA, polyA site and three prime non-coding regions One or more other factors such as range? Transformation by using a vector containing be done. [Clark, S, C, et al., Proc, Natl, Acad, Sci, USA, 81:2541-2547 (1984); Also, Kaufman, R, J, Proc, Na tl.

Acad, Sci, USA, 82: 689-693 (1985)'t'' It was done. ] These can be obtained from natural sources or synthesized by known methods. be done. Basically, various components present in DNA, such as virus functions, If available in large quantities or if they can be synthesized like poly A sites In some cases, large-scale vectors can be obtained by simply culturing a microbial source and extracting all of its DNA from suitable endonucleotides. The DNA was digested with Rease, the y-fragment was separated, and the DNA containing the desired factor was separated. By using appropriate restriction pixels, we can identify the DNA’ii and recover it. can get.

Various vector systems, such as polyomavirus and retrovirus Kanakura, are whether they are expressed by endogenous ADA-containing cells or exogenously expressed at higher levels It can be used if ADA is expressed from the ADA gene. Preferably 5 times or more It is desirable to have an expression of 10 times or more, more preferably 10 times or more.

Two types of vectors can be used for transformation here. Lid on non-bond One <lid> containing the exogenous ADA gene and the intended exogenous product gene. Transformation with 1-') and 1-') containing 1-' and 1-') can be performed at the same time. D.N. Methods for promoting cellular uptake of A are well known to those skilled in the art. quite good Transformation efficiency is determined by adding ADAll to the gene in molar excess of the product gene (preferably 10:1 or higher).

In order to achieve the most efficient simultaneous increase of ADA and product genes, the ADA gene It is preferable to use a combination vector in which the vector and the product gene are covalently linked. A The coding strands of the DA gene and the product gene are preferably connected to the stop codon of the product gene. 1 by direct ligation adjacent to the start codon of the ADA gene.

Even if these genes are linked through oligodeoxyribonucleotide columns, good. This oligonucleotide 5-bridge does not contain stop or start codons and R Palindromes (palindromes) to reduce the possibility of forming NA hairpin luzo structure) should not be included. Also, one or It is also possible to transform with two or more vectors.

Vectors used in producing cells or cell lines useful in the method of the invention are preferably is a helical, double-stranded, circular construct obtained from standard prokaryotic cloning methods. vector. However, (such as ligation to genomic auxiliary DNA) Even if the vector is a linear vector in which the covalent bond has been broken at one point during other steps, good.

One suitable vector is Park Loan 9 Ripe, Rockville, Maryland. ATCC deposit number 12301 American Type Culture Collection No. 39754 as Escherichia coli (E.

plasmid 'p91023 (B) deposited in MC1061 (coll) . This deposited vector was digested with EcoRI to delete the C3F' gene and It is modified by replacing it with the ADA gene. p91023(B) CH To fully express ADA″ in OMB cells and heavy hamster kidney cells (BHK) It was used.

In one embodiment of the present invention, a foreign ADA gene and a target protein are encoded. Polyoma replication origins and transcriptional enhancers are associated with foreign genes. Vectors are provided that contain. For example, the p91023 vector was known in the field Using technology, the 5V4Q enhancer is deleted, which causes horiomas and ADA. and modified by substitution with protein coding sequences. after that , this vector was used to induce s lioma-transformed mouse cells expressing high levels of T antigen. introduced into the cell line.

This polyoma system has considerable advantages, but is similar to that used in the CO8 system. are doing. GO8m cells are monkey kidney cells transformed by SV40; expresses the T antigen from 5V4Q. Plasmid total CO containing 5v40 origin of replication When introduced into 8#I cells, T antigen acts on its 5v40 origin of replication, resulting in a very high Special edition Showa 62-502662 (5) The number of copies of the plasmid will be replicated. Plasmids are thus high copy -number (approximately 50,000 copies/cell), so the cells die rapidly. , they are cultured for no more than two weeks.

Polyomas replicate with a sharper copy number than the CO8 system, which allows the cells to survive. Provide better condition for all items. The mouse cells in which polyomas can replicate are selected for full expression of T antigen from the oma. ADAi code and polio Mouse cells transformed with a plasmid containing a polyoma origin of replication. will be introduced in Replication occurs as a plasmid rather than by integration; The copy number is between 51,000 and 10,000 per cell. Uses polyoma cell bodies However, it is highly recommended for acF2 in the presence of high levels of adenosine or Xy1-A. By amplifying the It would be possible to obtain twice as high resistance to aCF.

In another embodiment of the invention, retroviral sequences and exogenous ADA genes are fully linked. A novel vector is provided. Group antigens, polymerases and The rope gene was deleted from the retrovirus, creating the ADA gene and the ADA gene. Substituted with appropriate transcription and packaging signals that enclose the offspring within the virus. It will be done. Such retrovirus construction techniques are known to those skilled in the art. Then this Viruses are transmitted from cell to cell. The presence of this ADA virus in other cells screened by selecting for the presence of increased ADA expression of . This vector has the ability to integrate the ADA gene into whole cells with very high efficiency. This is particularly desirable. Copy number increases after initial infection due to the presence of the ADA gene be done. This type of retroviral vector generates all cell lines useful in this method. Not only for infection, but also for infecting cells in ViVO in gene therapy of mammals. used to make

Once the host cell or cell line is injected with exogenous ADA DNA and the target protein, transformed with a vector containing a foreign gene encoding the desired transformation. Once cells are selected, they are tested for the linkage of product genes to their chromosomes. , or screened for expression of the product itself. raw material that can be used The product genes are essentially unlimited. Cells of higher animals such as moaning mammals and crawling animals Currently, the genes for active proteins and enzymes present in It is a gene that

The toxin has an adverse effect on all cells by hydrolyzing total synthesized dihost proteins. Even the genes for these proteins can be modified by adding antitoxins to the culture medium or reducing optimal expression levels. You can also use it by changing the method completely, such as selecting a lower level. Wear.

Screening for product gene linkage was performed using Southern blot analysis. be exposed. Screening for product expression can be performed using standard immunoassays, biochemical Assays or enzymatic assays can be utilized. Once the transformed cells are Once determined, the expression of the product gene can be a constant or increasing amount as described above. can be amplified by subculture in the presence of a selective agent. current By the way, it is preferable to use the 1l-AAU method with increasing concentrations of act'. Ru. Generally this means that (a) they preferentially produce products when compared to other cells in the cell population; one or more expressing cells are selected from the transformed cell population; (1)) selection; The selected cells are then subjected to conditions designed to select for changes in phenotypic expression. (C1 when compared to other cells in the subsequent population) one or more cells that preferentially express the product from its subsequent cell population Involves further selection. Step (b) advantageously comprises multiple clones of step (a). This is done using

Although all of the above methods can be used for both selection and amplification of transformed cells, In a preferred embodiment, a combination of different methods should be utilized. The A method is more sensitive than the 1l-AAU system in selecting for the uptake of exogenous DNA. seems to be good and consistent. Amplification of transformed cells is preferred is performed using the 11-AAU selection method.

Transformed cells can grow in any medium, but depending on the specific medium used, as described below. Some precautions must be taken depending on the law.

For example, fetal bovine serum contains higher levels of endogenous ADA than horse serum. X In the yl-A selection method, 3 nM aCF was used in the presence of 4.0 μM Xyl-A. In contrast, in the 1l-AAU selection method, 0.01 μM aCF is Used with 0.03 μM aCF in the presence of Adeno7ine. Therefore, extremely The selection method requires only low levels of cytotoxic agents (e.g. Xyl-A). If used, media containing high levels of endogenous ADA (e.g. fetal bovine serum) are can detoxify cytotoxic agents. If the use of fetal bovine serum is desired, an example of how to select For example, by switching to a different system, such as 1l-AAU, which uses significantly higher amounts of cytotoxic agent, Minimize the effects of fetal bovine ADA. You can also use another selection marker. You can also

If it is desired to use the X71-A selection method, a number of strategies can be used to problem can be overcome. Horse serum contains high levels of endogenous ADA'i. Therefore, it will be used in place of fetal bovine serum. However, fetal bovine serum If the use of fetal bovine serum ADA is desired, use a high concentration of effects can be minimized. Furthermore, just before selection, add X71-A2. or continue adding Xyl-A2 periodically to detoxify with fetal bovine ADA. X71-A > Can be replenished.

The following examples demonstrate the use of the method of the invention.

ADA cI) NA for expression was selected from previously published human and murine sequences. selected. For example, mouse ADA cDNA, pADA5-29 (Yun et al., supra) References 15179-15185] is a mammalian expression vector p90123 (C The 8F gene was induced from p91023(B) by deletion by 6EcoR engineering. guided). 1056 nucleotides in pADA5-29 The reading frame was excised by Nco engineering and EcoRI digestion. . Both ends were duplicated with the Flenow fragment of DNA polymerase l to make blunt ends. and ligated into the EcoR site of vector p91023. the obtained vector - p9ADA5-29 (see Figure 1) is (from left to right) an adenovirus. VA gene (VA), 72bp F i-7'! 5v40 replication origin containing j-f, Adenovirus Contains an Adenovirus Trisegment Leader and 5' Splice Site major late promoter (AaMLP), 3' splice acceptor site (3'ss ), ADA insert (ADA), dihydrofolate reductase insert (DHPR), 5V 4Q early p17A site (sv40) and p required for growth in E. coli Contains the BR322 sequence.

Using the vector p9ADA5-29, p cO8-1 was transfected. [Karatsumay (Kaufman, R, J, ), Proc, Natl, Acaa, Sci, USA, See above. [Transfected cells]9 The cells exhibit high levels of ossevation. The tick mouse received a zymogram showing that it produces ADA2. .

DHFR missing McHOmll, CHODHFR (DUKXBII) U10μ9/r in alpha medium containing rtl thymidine, deoxyadenosine and adenosine. Proliferated. These cells tKaufman et al. J, Mo1.　Bi, ox,, Yu mutual pADA5-29 (25μ g/10 cells) in trans;yzri)t, 7'c. Transfection 4 After 8 hours, (1) 10μ97ml thymidine, 15μ9/E hypoxanthine, 4 alpha medium containing μMXy1-A and various concentrations of aCFy2, or ( 2) 10 μg 7d thymidine, 10 μ9/rttl deoxyadenosine, 1 m A solution containing M uridine, 1.0 mM adenosine, and various concentrations of dCF t- The above cells were cultured in Lufa medium (8×10 cells/10 cm plate). both Four plates of each aCF' concentration were prepared for the medium. 2 used The media correspond to the Xyl-A selection method and the modified 11-AAU selection method, respectively.

The 1l-AAU method uses CHODHFR-cells (which can produce y4) I changed it because there is no need to use alanosine. endogenous to fetal bovine serum To avoid cytological agents and detoxification due to low rail ADA, 10% fetal bovine serum was added to the medium immediately before use.

This transfection method also used mock-transfected C for comparison. Make HODHFR-cells (do not insert foreign ADA DNAi into CHO cell line) repeated exactly as above. The results of the selection method are as follows: showed that the 1l-AU method was more sensitive in indicating the uptake of exogenous DNA. did. Selection for DNA uptake K is preferably about 4 μM Xy1-A and about 0 ．． 003-0.014M aCF'1: Measured using UB.

The transformed cells are described in the above-mentioned documents 8338-8345 by Yeung et al. and as previously modified with the exception of alanosine. The amplification was performed using the 11-AAU method using additional amounts of aCF2. Transformed cells are 10 Contains % Fetal Bovine Serum (Grand Island Biological Company) The ink was poured into DMEM at 37°C. Transformed CHODHFR-cells are It was possible to grow in the 1l-Au medium described above.

selected by 1l-AU selection at aCF concentrations of 0.03 μM and 0.1 μM Six transformed colonies were plated on the above medium. These cells then i0.1 were exposed to μM or 0.5 μM aCF, respectively. Don't produce large quantities of ADA2. The cells died. When the surviving cells begin to proliferate again, these cells become the same Passed several times through level aCF. Thereafter, the concentration of aCF was increased.

Cells were tested at 0.03 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM and 20 Exposure to aCF was performed stepwise in μM.

Cells to be sorted were harvested for one week after drug selection, and 10 cells were harvested 24 hours before harvest. Fresh MEM containing serum was added. Harvest cells by trypsinization, ・Wash three times with balanced salt solution of Nx (no Mg” and Ca), and Homogenization medium (10 mM) in a volume twice the filling volume of Lith-H (J.

PH7-5+ 1mM β-mercaptoethanol and 1mM EDTA ). Suspended hard pellets are frozen at -20°C, thawed, and then It was homogenized using a Ron homogenizer.

Sample 1’! Cell debris was removed by centrifugation twice at 15,000×9 for 30 minutes each. Supernatant( (containing approximately 1 ml of protein) was added directly to the starch gel. electrophoresis is It was carried out at 4°C for 16 hours at 200v or 5 hours at 400v. After electric flame motion, The starch gel was cut into replica sheets with a thickness of approximately 1 tone, and icilano, M, J.) O/ROMatography and electrophoresis (Smith) , 1. m collection) 4th edition, vol. 2, pp. 185-209, Wm.

As described in Electrophoresis Handbook, North/Haurant, and Oxford (1976). Histochemical staining was performed for azone sindeaminase activity as described.

For transformed cells selected with this treatment ID of 0.1 μMaCF, approximately 10 Double amplification was performed on the selected transgenic surface pe11 with 0.03 μM aCF'. This resulted in approximately 50 times amplification of iri. Further amplification is done using dc as described above. It is obtained by increasing F2 stepwise and continuing to apply selective pressure to viable cells.

Plasmid 9ADA5-297 described in Example 1, in place of the C3F' gene p91023 (containing the entire DNA sequence encoding the target product r'IJs peptide) B) Mixed with derivative p91023-p.

50 μ9 p91023-p total 0.5 μg mixed with pgADA5-29, Na 0Ac　(p)14. s) In addition, 0.3M and 2.5 times the capacity of ethanol Le? Precipitate by adding 9. The precipitated DN A was dried completely naturally, then 2 X HEBSS (0,5m/') (Chu et al., 3 holes 1 ．． Yu 3: I97-202 (1981) and resuspended in Kaufuma. Ng et al. J, Mo1. Biol 0, 25 M CaC0 as described above , 2 (0.5 mB). Phosphate calcium-DNA precipitate Chain 7 ( Chasin et al., Proc.

Natl, Acad, Sci, USA Near: 4216-4220 (198 1)a:Reference]. Kaufman et al. J, MOI, for the growth and maintenance of these cells. It is described in the above-mentioned publications of Blol, Id. and Cheyne/et al.

DUKX-B engineered cells were grown in 5 x 10/10 cfn dishes prior to transfection. The cells were cultured for 24 hours. Remove the medium and place the DNA-calcium phosphate precipitate in a monolayer. cells. Room temperature - 11" After 30 minutes incubation, 10 days fetal blood Add alpha medium (Flow) 5aI containing purified gold and incubate the cells at 37°C for 4.5 hours. Incubated. Then remove the medium from the monolayer of cells and add 10 tiglycerol. Alpha medium (flow) 2aI containing cells were washed and treated with 10 μl of fetal bovine serum, 10μ Gin, adenosine, deoxyadenosine, 4-nidyline and streptomycin Alpha medium containing was added. After 2 days, cells were incubated 1:15 in selection medium as above. subcultured.

Colonies will appear 10-12 days after subculture on selective medium. Select Two schemes are then performed: In the first plan, a single independent clone isolating transformed cells based on the uptake of exogenous ADNA, Each clone is then grown under conditions that increase the total expression of the product gene, i.e. The cells are grown in the presence of dCF at a concentration of dCF. In the second design, a large number of independent transformations A pool of cells is isolated and produced based on the uptake of exogenous ADA DNA. Under conditions that enhance the expression of biological genes, i.e. in the presence of progressively increasing concentrations of aCF. Propagate with. Individual clones are then isolated from mass-selected populations and the product remains Analyze gene expression. Clones with the highest levels of product gene expression , under conditions that further enhance product expression, i.e., gradually increasing the concentration of (IcF) in the medium. and multiply it.

Another method for amplifying the ADA and product gene kliJ is Unbound vectors p91023-p and p9ADA5 used in the method of this example p91023 vector containing both the ADA gene and the product gene instead of -9 Selection of heterologous ADA genes in mouse fibroblasts using pADA5-29 At the 5v4Q origin and transcriptional enhancer of 11P, polyomavirus Plasmid containing origin of replication and transcription enhancer)'pXc-ADA It was derived from this method.

Contains polyoma regulatory region linked to Xhol IJ anchor in BCI I part Starting plasmid p, 84. A2. X (Veldman et al., Mo 1. Ce1l Biol, 5:649-658 (1985) kg ” digested with limited endonuclease Bgll. 100 μM-f dAT at the end P, d, TTP, d, CTP and UaGTP (KT4 DNA polymer in the presence of D) In the repair reaction using Lase 1, the ends were blunt-ended.

(See Maniachu et al. above and it). EcoR Nilinka (Collaborative Lisa) This DN A f, r was digested with excess EcoRI and Xh, o. did. The obtained DN A was purified using Tris-boric acid as a buffer system. Fragments subjected to electrophoresis on a polyacrylamide gel and run at 370 bases was isolated by electroelution (step d).

The 370 bp fragment was Y-whitened in advance with XhoI and EcoRI, and BloI, (see above). The obtained plasmid was erased with Xho engineering and (Ja engineering). By converting the entire approximately 400 bp fragment into free sasu! :the law of nature. EcoR engineering part and C This fragment containing 24 bp'i from pBR322 between the xaI sites was isolated and It was ligated to pADA5-29, which had been previously digested with oI and C1aI. child E. coli HBIOI was transformed into tetracycline resistant using the DNA of Ronnie p, 84. A2. The original XhoI-Bgl fragment derived from X Filter hybridization to probes prepared by integration [Grunstein et al., Proc. Natl. Aca. d, Sci., 72:3961 (1975)'i]. I clicked. Positively hybridizing clones were analyzed and plasmids) ``pXC-ADA is made into a DNA1 band twice in u-1=sium. Prepared. The structure of the plasmid pXC-ADA was created after digestion with multiple restriction enzymes. This was confirmed by analysis.

pXC-ADA is described in Kaufman et al., Mol Blox, supra. Next mouse fibroblasts are transformed with the origin-deficient polyomavirus early region as (MOP, New York Medical College's C1audio Basilico (C1audio B) A day 111co) was used to transfect . However, the cells were grown in DME medium containing 10% fetal bovine serum.

Three types of transformation that induce a transformed phenotype in the early stages of polyomaviruses Expresses seven different antigens (canine, medium, and small T antigens).

ry,+)oma replication origin-containing protein introduced into mouse fibroblasts into canine T antigen. Induces Sumid replication. [Tynda'll et al., Nuc, A See c1ds Res, 9:6231-6250 (1981). 〕to 48 hours after transfection, cells were treated with acF4 with increasing concentrations of acF. μM Xyx-A f: Subcultured at 2 x 10 cells/dish in medium containing fca concentration Five plates were used for each test.

Two weeks later, cells transfected with pXC-ADA and mock transfected Both cells (no foreign DNA = i) were selected with 0.01μMaCF. had colonies when selected.

At 0.03μMaCF, 43 colonies appeared in the transfected cells. compared to 3 in mock-transfected cells. transfer This number in the 7′CI#U cell was o, iμMdCF was 34, and 0.3μ The number decreased to 15 with MaCF.

Mock-transfected cells showed no colonies at these high levels. won. The growth of cells at these high aCF concentrations resulted in transfected cells plasmid 'p without amplification by sequential selection at even higher aCF concentrations. It has been shown to contain multiple copies of Xc-ADA. pXC-ADA 'i envoy The selection for high levels of ADA expression in polyoma-transformed fibroblasts used for Resulting from high plasmid replication driven by polyoma replication signals Will.

Retroviral vector pEVX (Kriegler et al. 1.9 (See all references in Dorie, U. 8:483-491 (1984)) by Moloney. y) Sequences of both leukemia virus and Harvθy sarcoma virus? I was guided. pEVX is a fully functional Molonei leukemia virus. packaging part of Harvey sarcoma virus while retaining the ring signal sequence. modified by deleting the position (Proc, Natl, Acad, Sci., 72:3961 (1975)].

The obtained plasmid) pFVXM (see Figure 2) is the long terminus of the virus. (LTR) and an internal polylinker for insertion of heterologous genes. So These include retrovirus group antigens (gags), polymerases (pox) and Contains no envelope (env) gene. The Bg1D site of this plasmid is unique. period, which can produce the protein encoded by the inserted sequence. ideal for insertion and subsequent expression of the protein.

Foreign American ADA is inserted O7t into pFVXM, pADA5-29=iEco Digested with R and Sac and treated with T4 DNA polymerase to make both ends completely blunt. Convert and set Bgllll linker (collaborative research). It was obtained by After Bg11 digestion and electrophoresis through agarose gel, ca. A 1.8 Kb band was isolated. This fragment was previously digested with Bgll. 'Connected to VXM. Nick-translated DNA fragment (pADA5-2 9. Colony hybrida to the original EcoR/Sac I fragment isolated from Dicolonies were selected by ization (see Grunstein et al., supra).

DNA is extracted from positively hybridizing clones using restriction endonucleases. Prepared by analysis. One clone, pRetro ADA 1-1, is transcriptionally activated. (Long Terminal Repeat of retrovirus used for initialization) (LT R) was found to contain ADA inserted in the appropriate orientation.

pRetro ADA 1-I DNA should be grown in E. coli HBIOI was prepared by Vant-9 and twice with cesium chloride. Using this DNA, we Fibroblast W2 cells [defective Moronei virus not packaged into mature pilions] including genome gold; Mann et al., Ce1l 33: 153-159 ( (1983)] was transfected. However, gag, 1) 0 1 and env polypeptide (required for virus production, pRetr.

ADA1-1) is expressed from the deleted genome. this These proteins have all the functions deleted in pRetro ADA 1-1. Enough to supplement. 2X10’/’2 cells pRetro ADA 1 48 hours after C aPO4-mediated DNA transfection with -1 259 , cells were subcultured in 4 μM Xy1-A containing 0.01 M aCFk.

Three colonies appeared from the cells that received DNA1, but they lacked DNA. were selected and analyzed for retroviral production of ADA.

Conditioned medium from 10 cells (1 iJ) was collected after 24 hours, filtered (0.2 μM 3T3 cells (2X10) in the presence of 8μ97ml polybrene after filter) 2 hours were added. The virus was then removed and the cells were fed with fresh medium. 48 After an hour, the entire surface of 3T3 cells was treated with 4 μM Xyl-A! Hi O, o1'!　 The cells were subcultured at 1=10 in a medium containing 3 pM of dCFf. after 14 days All colonies were counted. Uninfected cells were collected at 2 x 10 initially infected cells 9 0.01 fc was 0.03 M. There were no colonies growing in aCF. It was. Infected cells were infected with approximately 4000 colonies with 0.01 μM aCF and 0.01 μM aCF. It had about 3000 colonies at 03 μM aCF'. These results are)1 Culture fluid from F2 cells transfected with 0 infectious units per liter shows that it existed.

This method uses a strong selection system to obtain cells expressing all the heterologous ADA. allows the introduction of amplifiable vectors.

It introduces all other genes into retroviruses using techniques well known in the art. However, it would also be possible to introduce them into whole cells. foreignness The presence of the ADA gene allows amplification of the inserted viral DNA. moreover, Amplification of the retrovirus sequence in v2 cells is necessary to introduce the entire gene into animals and humans. resulting in a higher titer of virus # needed for

pFVXM Hθ2 international search report

Claims (13)

[Claims] 1. at least one copy of an endogenous gene encoding ADA; an amplified copy of the foreign gene that encodes the protein and encodes the selected protein. selection, consisting of culturing cells containing amplified copies of a foreign gene that A method for expressing foreign proteins at high levels. 2. A cell containing an endogenous gene encoding ADA is transformed into a cell containing an endogenous gene encoding ADA. The gene and the exogenous gene encoding the selected protein are transformed and then co-amplifying the foreign ADA gene together with the foreign protein gene. The method according to claim 1, comprising: 3. A single expression vector containing a foreign protein gene and a foreign ADA gene 3. The method of claim 2, which comprises transforming said cells. 4. A single protein in which a foreign protein gene and a foreign ADA gene are covalently linked The method according to claim 3, which comprises transforming said cells with an expression vector. Law. 5. A first expression vector containing a foreign ADA gene and a foreign protein gene transforming said cell with a second expression vector comprising a second expression vector The method described in Section 2. 6. The cell is selected from the group consisting of yeast cells, bacterial cells and mammalian cell lines. , the method according to claim 1. 7. Mammalian cell lines include Bowes cell line, mouse L cell, and mouse line. Fibroblast cells, mouse MIH3T3 cells, human liver cancer HepG2 cell line and CHO 7. The method according to claim 6, wherein the method is selected from the group consisting of cell-like cells. 8. A cell line containing an endogenous gene encoding ADA is transformed into a cell line containing an endogenous gene encoding ADA. The gene and the exogenous gene encoding the selected protein are transformed and then simultaneously amplifying the foreign ADA gene and the foreign protein gene. microorganisms for high-level expression of selected foreign proteins. Cell strain. 9. Foreign genes encoding ADA include murine ADA, human ADA, and bacterial ADA. and yeast ADA. 10. The exogenous gene encoding ADA and its inclusion in the virus A vector containing retroviral transcription and package Nog sequences. 11. Claim 1 further comprising a gene encoding a foreign protein of interest Novector described in item 0. 12. Foreign gene encoding ADA and gene encoding target protein Progeny: adenovirus VA gene, SV40 replication origin, adenovirus major vector containing in association with the early promoter and the SV40 early polyA site. 13. Foreign gene encoding ADA and gene encoding target protein The offspring are associated with the polyomavirus origin of replication and polyomavirus transcriptional enhancer. Vectors containing in association.